Background. Recently, we and others have characterized a significant population of tumor- associated leukocytes which co-express endothelial and myeloid markers, the so called "vascular leukocytes". Most studies indicate that they serve as potent mediators of tumor growth and angiogenesis. Generation of vascular leukocytes is likely modulated by cues from the tumor microenvironment. The mechanism of their generation is not known. Objective/Hypothesis: Our preliminary data suggest that TNF results in a dose-dependent upregulation of endothelial-specific markers in cultured monocytes. This effect was dependent on TNF receptors as cells isolated from mice lacking TNF receptors displayed dramatically delayed kinetics in myeloid/endothelial biphenotypic cell generation. Mice implanted with TNF overproducing tumors contained larger, more vascular tumors with increased numbers of vascular leukocytes than contralateral, control tumors. Based on these exciting data, we hypothesize that locally-derived TNF is an important regulator of tumor growth by regulating, in part, the generation of myeloid/endothelial biphenotypic cells. Specific Aims: 1) Determine the role of TNF in vascular leukocyte formation in mouse models of tumor growth by upregulating local tumor-derived expression and by immunoblocking approaches with bone marrow transplant models. 2) Delineate the mechanism(s) by which TNF promotes vascular plasticity of leukocytes. Relevance to VA mission: Cancer is a leading cause of morbidity and mortality among our aging Veterans. This grant is based on preliminary data that support a novel hypothesis that the "proangiogenic" and tumor-promoting properties of TNF are mediated, at least partly, by facilitating myeloid to endothelial differentiation that contributes directly to specific proangiogenic vascular leukocyte subpopulations within both breast and melanoma skin cancers. This work may lead to the use of TNF inhibitors as adjunct therapy to manipulate specific myeloid populations to abrogate tumor progression in our Veterans. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE. It is widely accepted that many aspects of cancerous growth is mediated by inflammation. Tumor necrosis factor-alpha (TNF) is an important mediator of inflammation. It was first isolated from the blood of mice suffering from severe microbial-induced inflammation and was shown to induce tumor necrosis or death (hence the name). Importantly, the doses of TNF used in such experiments were very large. Endogenous TNF, however, is produced at chronic, low levels in tumors. Several recent studies suggest that at these low, chronic doses, TNF promotes tumor growth. For example, mice that have a deletion in TNF gene or the machinery by which it acts, have delayed tumor growth and metastasis. However, there is a gap in our understanding on how TNF serves as a tumor promoter. Our laboratory has long studied the role of blood vessel stem cells in cancer. We and many others have shown that myeloid lineage cells can upregulate endothelial markers in vitro and in vivo. These myeloid/endothelial biphenotypic cells represent a subpopulation of myeloid cells that are promote tumor growth and blood vessels. Using tumors that express slightly higher levels of TNF, we have strong evidence that at least one important mechanism by which TNF works within tumors is by promoting the differentiation of myeloid/endothelial cells. These findings are entirely novel and have important implications in cancer therapy. TNF inhibitors are already available and FDA-approved to treat inflammatory disorders, such as arthritis. By understanding the details of TNF's role in tumor- myeloid cell interactions, we may be able to develop new cancer therapeutic strategies using TNF-inhibitors. Cancer continues to be a pervasive and prevalent health problem among our aging Veterans and critical to the biomedical research mission of the Veterans Affairs.